HISTOCHEMISTRY OF MYELIN—XII ANIONIC STAINING OF MYELIN BASIC PROTEINS FOR HISTOLOGY, ELECTROPHORESIS AND ELECTRON MICROSCOPY

Abstract— Phosphotungstic acid haematoxylin, trypan blue and amidoblack techniques have been developed as anionic dye methods for staining myelin basic proteins. All methods displayed central and peripheral nervous system myelin in histochemical prepa rations and stained brain basic proteins in electrophoretic polyacrylamide gels: phosphotungstic acid haematoxylin appeared to be the most selective of these techniques. Electron photomicrographs of peripheral nerve stained by phosphotungstic acid haematoxylin showed that the major part of myelin basic protein is located in the period dense line. The basic proteins stained by phosphotungstic acid haematoxylin showed an early loss in rat sciatic nerve undergoing Wallerian degeneration and had completely disappeared from the centre of 20 plaques of multiple sclerosis.     

HISTOCHEMISTRY OF MYELIN–XV. CHANGES IN THE MYELIN PROTEINS OF THE PERIPHERAL NERVE UNDERGOING WALLERIAN DEGENERATION–ELECTROPHORETIC AND MICRODENSITOMETRIC OBSERVATIONS

The chronological order of changes in rat peripheral nerve proteins during Wallerian degeneration has been investigated by microdensitometric and electrophoretic techniques. Both methods revealed an early loss of myelin proteins. The histochemical microdensitometric study showed a very substantial early loss of stainable protein basic groups and a somewhat slower progressive loss of the major protein component of peripheral nerve myelin (the J band). The electrophoretic study showed an early loss of both the J band protein and the slower-moving basic protein band. The histochemical study also suggested that some cerebroside may be lost in the early stage of Wallerian degeneration. It is concluded that degradation of myelin proteins is an initial event in the process of myelin breakdown.     

PROTEIN COMPOSITION OF MYELIN OF THE PERIPHERAL NERVOUS SYSTEM

Abstract— Myelin was purified from the peripheral nervous system (PNS) of several species. The protein composition of these preparations was examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Proteins characteristic of all samples include, in order of increasing mobility: a series of high molecular weight proteins, the major peripheral nerve protein (P₀), two uncharacterized proteins, and two basic proteins (P₁ and P₂). Quantitative results, obtained by densitometry of gels stained with Fast Green showed differences in protein distribution, both between species, and from different types of nerves obtained from the same animal. The relative amounts of P₁ and P₂ proteins were the most variable; e.g. myelin from guinea-pig sciatic nerve had little or no P₂ protein, whereas 15 per cent of the myelin protein of beef posterior intradural root was Pz protein. P₀, P₁ and P₂ proteins from rabbit sciatic nerve and P₀ and P₂ proteins from beef dorsal and ventral intradural roots were purified and their amino acid compositions were determined. Our results indicated that the P₁ protein is very similar in size and amino acid composition to the basic protein of central nervous system myelin, whereas the P₀ and P₂ proteins are unique to the PNS.     

COMPARATIVE STUDIES ON THE MYELIN PROTEINS OF BOVINE PERIPHERAL NERVE AND SPINAL CORD

Abstract— (1) Two myelin fractions of bovine peripheral nerve and spinal cord have been studied comparatively. Cholesterol as well as cerebroside content per mg of protein in the peripheral nerve myelin was less than that in the spinal cord myelin, while no significant difference in the total phospholipid content was noted.
(2) The basic proteins in myelin fractions were quantitatively estimated by disc gel electrophoresis. Around one-fourth of the total myelin protein in the bovine peripheral nerve was a basic protein with a mobility of 1.07 relative to lysozyme by Reisfeld's disc gel electrophoresis.
(3) The myelin proteins in the peripheral nerve were less completely solubilized than those of the spinal cord by treatment with deoxycholate as well as by Triton-salt solution. The protein fractions obtained from the peripheral nerve myelin by techniques similar to that for obtaining the proteolipids from the spinal cord myelin, contained different types of protein.
(4) 2',3'-Cyclic nucleotide 3'-phosphohydrolase activity in the peripheral nerve myelin was only one tenth of that in the spinal cord myelin. The Triton-salt insoluble fraction showed remarkable high activity among subfractions of the spinal cord myelin.
(5) By immunological studies, it may be concluded that an antigenic substance for experimental allergic neuritis was localized in the peripheral nerve myelin, but not in its basic protein.     

ASSOCIATION OF AXONALLY TRANSPORTED PROTEINS WITH GOLDFISH BRAIN MYELIN FRACTIONS

—The presence of rapidly transported axonal proteins in purified preparations of myelin has been investigated in the goldfish visual system. Fish were injected intraocularly with ³H proline and contralateral optic tecta were pooled 8–12 h later for purification of myelin. Three purification procedures were employed using continuous and discontinuous gradients of sucrose and continuous gradients of CsCl. All of the myelin preparations were found to have physical, chemical and enzymatic properties attributable to relatively pure preparations of myelin. The goldfish myelin differed from mammalian preparations in having a slightly lower density and in containing an additional major protein of approx. 45,000 mol. wt. All of the myelin preparations retained relatively high levels of axonally transported radioactivity with specific radioactivities which ranged from 70 to 80 per cent of that of the whole tectal homogenate. Acrylamide gel analysis showed the myelin-associated radioactivity to be confined to the higher molecular weight proteins with very little radioactivity associated with basic protein or proteolipid protein. Both the axonally transported radioactivity and the group of higher molecular weight proteins were found to be more concentrated in a myelin subfraction of relatively high density than in a subfraction of low density. The possible significance of the association of axonally transported proteins with myelin is discussed.     

Production and Characterization of Monoclonal Antibodies to Peripheral and Central Nervous System Myelin

Monoclonal antibodies against P0, myelin basic protein, or myelin-associated glycoprotein were generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with central and peripheral nervous system myelin proteins. The antibodies secreted were either IgG, IgM, or IgA. Clone C6B5 (iso-type IgM) secreted antibody(ies) that bound to both myelin basic protein and myelin-associated glycoprotein, although binding of antibody to myelin basic protein as detected by the immunoblot technique appeared to be much less than to the myelin-associated glycoprotein. Antibodies were characterized in solid-phase radioimmunoassay for their species cross-reaction, and histologically for the specificity of binding to myelin in central and peripheral nervous system tissues. These monoclonal reagents should prove valuable in studying CSF and myelin-producing cells, since in both cases the concentration of myelin proteins is low.     

Basic proteins of rodent peripheral nerve myelin: immunochemical identification of the 21.5K, 18.5K, 17K, 14K, and P2 proteins

Abstract: Proteins in peripheral nervous system and central nervous system myelin and homogenates of sciatic nerve and brain from young and adult mice and rats were characterized with affinity-purified anti-P₂ and anti-myelin basic protein sera after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Using this method we have identified a component of rodent peripheral nervous system myelin as P₂ protein. Peripheral nervous system myelin also showed the presence of four basic proteins in addition to P₂ protein. These were found to be analogous to the 14, 17, 18.5, and 21.5K species found in the central nervous system myelin. A number of high-molecular-weight proteins were also detected with anti-myelin basic protein serum in peripheral nervous system, as well as central nervous system myelin. In addition, we report the presence of a high-molecular-weight P₂ cross-reactive protein in rodent brain stem homogenates, but not in central nervous system myelin.     

QUAKING MOUSE: ISOLATION AND CHARACTERIZATION OF MYELIN PROTEIN

A new technique, involving final purification on a continuous CsCl gradient, was utilized for the isolation of cerebral myelin from adult (4- to 6-month-old) quaking mice, littermate controls and young (10-day-old) normal mice. The yield of myelin from either adult quaking or normal young mice was 5-10 per cent of that from adult controls. After deli-pidation, myelin proteins were separated by polyacrylamide gel electrophoresis in buffers containing sodium dodecylsulphate. Two gel systems were utilized: (1) a high-resolution discontinuous electrophoresis system; and (2) a continuous system utilizing gels cross linked with ethylenediacrylate (EDA). The gels from the discontinuous system were stained with Fast Green and quantified by densitometry. The base lability of the EDA-linked gels permitted direct chemical determination of protein in specific bands. Myelin from brains of normal adult mice contained, as major components, one proteo-lipid and two basic proteins. There were also a number of high-molecular-weight proteins which represented a significant portion of the total. Myelin from quaking mice had qualitatively a similar distribution of proteins but the high-molecular-weight fraction comprised a much greater percentage of the total protein. The ratio of basic to proteolipid protein in preparations from quaking mice was considerably higher than that in the myelin from control mice. The distribution pattern of the myelin proteins from 10-day-old mice was quantitatively similar to that of quaking mice. Altogether the evidence supports the hypothesis that the quaking mutant provides a model of an immature nervous system with respect to myelination.     

PROTEIN COMPOSITION OF AXONS and MYELIN FROM RAT and HUMAN PERIPHERAL NERVES

Abstract— Proteins of rat and human peripheral nerves were studied in whole nerve homogenates and in purified myelin and axonal preparations of peripheral nerve. Both myelin and axonal fractions were obtained from desheathed and minced nerve segments by flotation and sedimentation, respectively, in 0.85 m -sucrose following hypotonic treatment. The purity of myelin and axonal preparations was confirmed by electron microscopic examination of pelleted material. Nerve proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 8.3 and 7.4. Major protein bands of fresh whole nerve homogenates corresponded to polypeptide bands of either the purified myelin or axon preparations. The most prominent electrophoretic band in peripheral nerve was identified as a myelin glycoprotein with molecular weight of 27,000. The major polypeptides of axon preparations had molecular weights of 200,000, 150,000, 69,000, 55,000 and 27,000. The latter two proteins were believed to represent tubulin and residual major myelin protein, respectively. The three largest axonal polypeptides were believed to be derived from neurofilaments, which represented the predominant organelle of the purified axons. Collagen was also seen in whole nerve homogenates and in purified axons but could be distinguished by its metachromatic staining with Coomassie blue.     

Partial deficiencies in the myelin proteins of developing mice heterozygous for the shiverer mutation

The central nervous system of the shiverer mouse is known to be severely deficient in myelin. Animals heterozygous for this autosomal-recessive mutation were crossed, and the myelin proteins were examined in the brains and spinal cords of shiverers and unaffected littermates among the offspring. In the brains and spinal cords of nine of the 14 unaffected littermates examined, the quantities of the myelin basic and proteolipid proteins were lower than normal. Furthermore, in the brains of heterozygotes 33 to ～ 150 days old, the myelin basic and proteolipid proteins were reduced in amount, compared to wild-type controls; the myelin basic protein was also present in subnormal amounts in the spinal cords from heterozygous animals at the ages of 17 to 150 days. More severe reductions in the quantities of the myelin proteins were observed in central nervous system tissue from homozygous shiverer mice, and the quantity of the myelin proteolipid protein in the central nervous system of the shiverer mouse, expressed as a ratio to the control value at each age, underwent a developmental decline. In heterozygotes, as well as shiverers, the peripheral nerves were also deficient in the P₁ and P_r proteins, which are the same as the basic proteins in rodent central nervous system myelin. The findings regarding heterozygotes suggest that the defective primary gene product in the shiverer mouse could be the myelin basic protein itself or a protein required for a rate-limiting step in the processing of the myelin basic protein.     

PROTEIN AND GLYCOPROTEIN COMPOSITION OF MYELIN AND MYELIN SUBFRACTIONS FROM BRAINS OF ''QUAKING'' MICE

Abstract— Quaking mutants in mice are known to be affected by an arrest of myelinogenesis and to have a purified myelin which is more dense than that of controls. Their myelin has been shown to demonstrate a striking decrease in proteolipid protein, a lesser decrease in the small myelin basic protein and changes in glycoproteins comprising reduction in the major peak and shift of this peak towards a higher apparent molecular weight. The possibility that these findings might reflect merely contamination of myelin with other membranes was tested by subfractionation. Light myelin (floats on 0.62 m -sucrose) is generally accepted as more compact and mature than the heavier subfraction (floating on 0.85 m -sucrose). The changes previously found were present in both subfractions and even more marked in the light myelin. These results indicate that the anomalies of myelin proteins and glycoproteins were not caused by contaminants and are present in compact myelin as well as in membranes which are transitional between the glial plasma membrane and the myelin sheath. Therefore, we suggest that the Quaking mutation results in dysmyelination rather than hypomyelination.     

THE SYNTHESIS OF MYELIN IN DEVELOPING RAT BRAIN

Abstract— —The synthesis of myelin proteins has been studied in the grey and white matter slices of developing rat brain by measuring the incorporation of [³H]lysine and [¹⁴C]arginine into polypeptide. The incorporation was sensitive to cycloheximide and puromycin at 1 mM concentration. Developing rat optic nerve slices, free of retinal ganglion cells, were able to synthesize myelin basic and proteolipid proteins, but rat retinal preparation failed to synthesize myelin basic protein. Rabbit retinae were able to synthesize myelin basic and proteolipid proteins. Significant activity of the myelin marker enzyme 2',3'-cyclic nucleotide-2'-phosphodiesterase has been found in the rabbit retina but not in rat retina. The results presented in this communication suggest that myelin proteins in the rat CNS are synthesized by the oligodendroglial cells and that neurons probably do not participate.     

MYELIN SYNTHESIS IN VITRO: A COMPARATIVE STUDY OF CENTRAL AND PERIPHERAL NERVOUS TISSUE

Abstract— Brain, spinal cord and sciatic nerve from rats at different ages were incubated for 2 h in a medium containing [¹⁴C]acetate and [¹⁴C]leucine as the precursors for synthesis of lipids and proteins. Myelin was purified from the incubated tissues and the specific and total radioactivites of myelin lipids and protein were determined. The uptake of radioactive precursors decreased with increasing age up to 6 months of postnatal age, the decrease following the same pattern for the three types of myelin. After age 6 months the uptake of the protein and lipid precursors reached a plateau that persisted up to 18 months, the oldest postnatal age studied. The amount of myelin isolated and the total myelin lipids extracted from both the central and peripheral nervous systems increased continuously from age 25 days to 18 months after birth. Consequently we suggest that myelination is a process that continues during the whole life of the rat.
The metabolic activity of peripheral nerve myelin was higher than myelin from the CNS at all ages studied. Although myelination in the sciatic nerve begins before that in brain and spinal cord, the three types of myelin apparently reach maturity at the same age. Lecithin exhibited the highest metabolic activity of the individual myelin lipids at all ages in both the central and peripheral nervous system. The metabolic activity of cholesterol in myelin from the 25-day-old rats was similar to that of lecithin but decreased to very low levels in myelin from the 18-month-old rats.     

THE OCCURRENCE OF TWO MYELIN BASIC PROTEINS IN THE CENTRAL NERVOUS SYSTEM OF RODENTS IN THE SUBORDERS MYOMORPHA AND SCIUROMORPHA

Extracts containing myelin basic proteins have been prepared from CNS tissue of representatives of the three suborders of Rodentia—Myomorpha, Hystricomorpha and Sciuro-morpha. Analyses of the extracts by electrophoresis at low pH showed that one type (L) of myelin basic protein is present in the CNS of all of the rodents examined (rat, mouse, hamster, guinea pig, chinchilla, prairie dog, woodchuck and squirrel). This protein is comparable in molecular size and charge to the CNS myelin basic proteins found in several other mammalian orders. In the CNS of the myomorphs (rat, mouse, hamster) and sciuro-morphs (prairie dog, woodchuck, squirrel) there is an additional type (S) of myelin basic protein of higher cathodic mobility and smaller molecular size. This additional protein is absent from the CNS of the hystricomorphs (guinea pig, chinchilla). These findings indicate that the presence of two myelin basic proteins originally reported in the CNS of the inbred rat is not an anomaly of inbreeding. These data further suggest that the presence of a single L-type CNS myelin basic protein might be a general characteristic of hystricomorphs, while the presence of both L- and S-type CNS myelin basic proteins might be a general characteristic of the myomorphs and sciuromorphs. Analyses by electrophoresis at low pH failed to reveal differences in mobility among either the L-type or the S-type CNS myelin basic proteins of the different species. In contrast, when electrophoresis was carried out cathodically at high pH, species-related differences in mobility were observed among the L- and S-type CNS myelin basic proteins.     

KINETICS OF ENTRY OF PROTEINS INTO THE MYELIN MEMBRANE1

Brain slices were prepared from 17-day old rats, and incubated with [³H]glycine or [³H]-leucine to label proteins. Myelin was isolated from the slices, and the proteins were separated by discontinuous gel electrophoresis in buffers containing sodium dodecyl sulfate. Radioactive basic and Wolfgram proteins appeared in myelin at similar initial rates, and their entry was nearly linear between 15 and 120 min with no detectable lag. Radioactive proteolipid protein appeared in myelin at one-fourth the rate of the basic and Wolfgram proteins between 0 and 30 min, then entered at a rate comparable to the other proteins between 45 and 120 min. When cycloheximide (0.2 mM) or puromycin (1.0 mM) was added, appearance of newly labeled basic and Wolfgram proteins in myelin stopped while proteolipid protein continued to appear in myelin at a normal rate for at least 30 min. Chase experiments with unlabeled glycine had similar effects. These results indicate the existence of a previously synthesized precursor pool of proteolipid protein with a 30-min interval between synthesis of proteolipid protein and its appearance in myelin. Incorporation of [³H]fucose into glycoprotein of the myelin sheath was studied, as was inhibition of incorporation of radioactivity by the use of either cycloheximide, or dilution with unlabeled fucose. The results indicated fucosylation of a sizable pool of presynthesized protein and a delay of 30 min between fucosylation of these polypeptides and their subsequent appearance in myelin as glycoproteins.     

STUDIES OF THE MECHANISM OF DEMYELINATION REGIONAL DIFFERENCES IN MYELIN STABILITY IN VITRO1

—Incubation of slices of rat central nervous system in Krebs-Ringer bicarbonate buffer produced a lipoprotein fraction which floated on 10·5% sucrose after homogenization of the slices and centrifugation. This fraction was not found after homogenization and centrifugation of fresh tissue and appeared to depend upon incubation. The amount of the light fraction increased in the following order per 100-mg slice: cerebrum < thalamic area < cerebellum < brain stem < spinal cord. The lipid composition of this fraction was similar to that of myelin, but contained a lower protein content compared to myelin of the corresponding area. This fraction was termed ‘dissociated myelin’. Upon incubation of slices a portion of the basic protein was lost from myelin subsequently isolated, and the dissociated fraction was slightly enriched in basic protein. The distribution of myelin protein among the characteristic three groups (basic, proteolipid and high mol. wt.) was quite different in myelin from spinal cord compared to that from other CNS area. Spinal cord myelin contained about 17% protein compared to about 23% in cerebrum, with brain stem myelin intermediate (19%), and the difference appeared to be due to lesser amounts of proteolipid in the caudal areas. The amount of dissociation after incubation was about 3–5 per cent of the total myelin in the cerebral cortex, 10 per cent in the thalamic area, 20 per cent in cerebellum, 35 per cent in the brain stem, and around 45 per cent in spinal cord. The smaller amount of proteolipid protein in spinal cord myelin may result in a deficiency of cohesive forces holding lipids and proteins together, thus causing greater instability and dissociation. Myelin dissociation increased with time of incubation up to 3 h, was augmented by Ca²⁺, and was substantial at pH 11, reaching a peak at pH 7, then decreased in the acid range. A similar fraction has been isolated previously from fresh CNS tissue made edematous by chronic treatment of rats with triethyl tin. The possible relationship of swelling in the disease process and myelin dissociation are discussed.     

THE PROTEIN COMPOSITION OF ISOLATED MYELIN1

—Three fractions, each containing markedly different proteins, was obtained from myelin: (1) The first fraction was obtained as an insoluble residue when myelin was extracted with neutral chloroform-methanol (CM, 2:1, v/v). It was digestible with trypsin and had an amino acid composition similar to that of the acidic proteolipid protein of Wolfgkam (1966). (2) The second fraction was obtained as a precipitate by the addition of various electrolytes (KCl, NaCl, CaCl₂, MgCl₂ or HCl) to the CM (2:1 v/v) extract. This fraction consisted mainly of a basic protein which exhibited an electrophoretic mobility and amino acid composition indistinguishable from those of the basic protein obtained from white matter (Martensson and LeBaron, 1966). This procedure provided for a simple and rapid isolation of the basic protein from myelin. Depending on the conditions of precipitation, this fraction was either free of lipid or contained tri- and diphosphoinositide. The effects of different ions at differing concentrations and the yield and nature of the precipitate have been studied. (3) A third fraction remained in solution in CM (2:1, v/v) after the addition of the electrolyte. It comprised the bulk of the myelin lipids and a protein fraction which was resistant to digestion with trypsin and had an amino acid composition similar to the classical proteolipid protein of Folch-Pi and Lees (1951). The possibility of a salt-type bonding between the basic protein and the polyphosphoinositides is discussed, and values for tri- and diphosphoinositide in bovine myelin are given.     

THE FORMATION AND STRUCTURE OF MYELIN SHEATHS IN THE CENTRAL NERVOUS SYSTEM

The development and structure of myelin sheaths have been studied in the optic nerves of rats and of Xenopus laevis tadpoles. Both potassium permanganate- and osmium-fixed material was examined with the electron microscope. In the first stage of myelinogenesis the nerve fibre is surrounded by a cell process which envelops it and forms a mesaxon. The mesaxon then elongates into a loose spiral from which the cytoplasm is later excluded, so that compact myelin is formed. This process is similar to myelinogenesis in the peripheral nervous system, although in central fibres the cytoplasm on the outside of the myelin is confined in a tongue-like process to a fraction of the circumference, leaving the remainder of the sheath uncovered, so that contacts are possible between adjacent myelin sheaths. The structure of nodes in the central nervous system has been described and it is suggested that the oligodendrocytes may be the myelin-forming cells.     

LOCALIZATION OF A BASIC PROTEIN IN THE MYELIN OF VARIOUS SPECIES WITH THE AID OF FLUORESCENCE AND ELECTRON MICROSCOPY

In this study, alanine was shown to be the N-terminal amino acid of a basic protein of low molecular weight that was isolated from either human or guinea pig brain. Antibodies prepared against the guinea pig protein were labeled with either fluorescein or ferritin. Studies with the labeled antibodies showed that an immunohistochemically similar protein is found in the myelin sheaths of central and peripheral nervous tissues of chicken and frog and a variety of mammalian species. Loss of integrity of the myelin during processing was shown to enhance markedly the antigen-antibody reaction.     

SOME PROPERTIES OF A MAJOR STRUCTURAL GLYCOPROTEIN OF SCIATIC NERVE

The major protein of rat sciatic nerve is a glycoprotein which consists of a protein moiety attached to galactose, mannose and perhaps other sugars. On controlled tryptic digestion, it splits into a glycopeptide and a smaller fragment similar in molecular size to peripheral nerve basic proteins. Both the basic proteins of peripheral nervous system (PNS) myelin and the glycoprotein are antigenically active when administered to guinea pigs and produce sciatic nerve lesions similar to those described for experimental allergic neuritis. It is suggested from the amino acid analysis and its antigenic properties that the protein moiety of the glycoprotein may contain a sequence which is similar to the basic proteins of PNS myelin.